Atrial natriuretic factor (ANF) is a polypeptide hormone synthesized and secreted by atrial cardiocytes in response to increased atrial pressure; it plays a major role in blood pressure and fluid homeostasis. Cyclic nucleotides have been implicated as second messengers mediating at least some of these effects. Cultured human fibroblasts used as a model system have been found to have a single class of high-affinity ANF receptor sites. Treatment with ANF caused a dose-dependent increase in cGMP. ANF also mediated a reduction in agonist-stimulated cAMP levels without effects under basal conditions. Pertussis toxin-catalyzed ADP-ribosylation of the Alpha subunit of Gi did not block the ANF-mediated reduction of cAMP levels: hence, Gi does not mediate ANF effects. The phosphodiesterase inhibitors IBMX, Ro 20-1724, and cilostamide did block the inhibitory action of ANF, from which we infer that the action of ANF may be mediated by the activation of a cAMP phosphodiesterase. The cGMP analogue, 8-Br cGMP, inhibited agonist-stimulated cAMP to a degree similar to that observed with ANF. The presence of 8-Br cGMP and ANF together resulted in no greater degree of inhibition. This suggests that the ANF-mediated reduction in agonist-stimulated cAMP may be caused by a cAMP phosphodiesterase, which was in turn activated by ANF-induced cGMP.